Craniofacial abnormalities in humans account for about 35% of defects in live births. Recent studies indicate that mutations in genes that play a central role in early placode development result in some craniofacial dysmorphologies. Placodal derivatives comprise important elements of the vertebrate head and developmental defects in these structures have devastating functional and psychological effects on individuals. Work in animal models demonstrates that Six1 is a key transcription factor that regulates the initial formation of the cranial placodes, and that Eya1 is a crucial co-factor for Six1 (reviewed in Brugmann and Moody, 2005;Moody, 2007). However, very little is known about the downstream targets of Six1 that are involved in craniofacial development. Furthermore, SIX and EYA mutations account for less than half of the cases of BO/BOR, indicating that mutations of yet-to-be discovered genes contribute to these craniofacial defects. I hypothesize that elucidating the direct downstream targets of Six1, which are thereby in the same regulatory pathway, will reveal additional causative genes for BO/BOR. I performed a microarray gene expression assay and identified a large number of potential Six1 target genes. I now propose to test whether these genes are involved in craniofacial development (Specific Aim 1), whether they are direct targets of Six1 (Specific Aim 2) and whether Six1 regulates them by transcriptional activation or repression (Specific Aim 3). These experiments will identify new genes involved in craniofacial development, and potentially elucidate the genetic network that underlies important craniofacial defects. Public Health Relevance: Craniofacial abnormalities in humans account for about 35% of defects in live births and recent studies indicate that mutations in genes that play a central role in early placode development result in the craniofacial deformation, branchio-otic (BO) and branchio-oto-renal (BOR) syndromes. However, Six1 mutations account for less than half of the cases of BO/BOR, indicating that mutations of yet-to-be-discovered genes contribute to these craniofacial defects. Employing a gene expression microarray assay, I identified a large number of genes that may act in the Six1 regulatory pathway and propose to test whether these genes are involved in craniofacial development, in order to elucidate the genetic network that underlies important craniofacial defects.